Multicellular organisms are dependent on advanced mechanisms of information transfer between cells and body compartments. The information that is transmitted can be highly complex and can result in the alteration of genetic programs involved in cellular differentiation, proliferation, or reproduction. The signals, or hormones, are often simple molecules, such as peptides, fatty acid, or cholesterol derivatives.
Many of these signals produce their effects by ultimately changing the transcription of specific genes. One well-studied group of proteins that mediate a cell's response to a variety of signals is the family of transcription factors known as nuclear receptors, hereinafter referred to often as “NR”. Members of this group include receptors for steroid hormones, vitamin D, ecdysone, cis and trans retinoic acid, thyroid hormone, bile acids, cholesterol-derivatives, fatty acids (and other peroxisomal proliferators), as well as so-called orphan receptors, proteins that are structurally similar to other members of this group, but for which no ligands are known (Escriva, H. et al., Ligand binding was acquired during evolution of nuclear receptors, PNAS, 94, 6803–6808, 1997). Orphan receptors may be indicative of unknown signaling pathways in the cell or may be nuclear receptors that function without ligand activation. The activation of transcription by some of these orphan receptors may occur in the absence of an exogenous ligand and/or through signal transduction pathways originating from the cell surface (Mangelsdorf, D. J. et al., The nuclear receptor superfamily: the second decade, Cell 83, 835–839, 1995).
In general, three functional domains have been defined in NRs. An amino terminal domain is believed to have some regulatory function. A DNA-binding domain hereinafter referred to as “DBD” usually comprises two zinc finger elements and recognizes a specific Hormone Responsive Element hereinafter referred to as “HRE” within the promoters of responsive genes. Specific amino acid residues in the “DBD” have been shown to confer DNA sequence binding specificity (Schena, M. & Yamamoto, K. R., Mammalian Glucocorticoid Receptor Derivatives Enhance Transcription in Yeast, Science, 241:965–967, 1988). A Ligand-binding-domain hereinafter referred to as “LBD” is at the carboxy-terminal region of known NRs. In the absence of hormone, the LBD of some but not all NRs appears to interfere with the interaction of the DBD with its HRE. Hormone binding seems to result in a conformational change in the NR and thus opens this interference (Brzozowski et al., Molecular basis of agonism and antagonism in the oestrogen receptor, Nature, 389, 753–758, 1997; Wagner et al., A structural role for hormone in the thyroid hormone receptor, Nature, 378, 690–697, 1995). A NR without the HBD constitutively activates transcription but at a low level.
Coactivators or transcriptional activators are proposed to bridge between sequence specific transcription factors and the basal transcription machinery and in addition to influence the chromatin structure of a target cell. Several proteins like SRC-1, ACTR, and Grip1 interact with NRs in a ligand enhanced manner (Heery et al., A signature motif in transcriptional coactivators mediates binding to nuclear receptors, Nature, 387, 733–736; Heinzel et al., A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression, Nature 387, 43–47, 1997). Furthermore, the physical interaction with repressing receptor-interacting proteins or corepressors has been demonstrated (Xu et al., Coactivator and Corepressor complexes in nuclear receptor function, Curr Opin Genet Dev, 9 (2), 140–147, 1999).
Nuclear receptor modulators like steroid hormones affect the growth and function of specific cells by binding to intracellular receptors and forming nuclear receptor-ligand complexes. Nuclear receptor-hormone complexes then interact with a hormone response element (HRE) in the control region of specific genes and alter specific gene expression.
The Farnesoid X Receptor alpha (FXR; hereinafter also often referred to as NR1H4 when referring to the human receptor) is a prototypical type 2 nuclear receptor which activates genes upon binding to promoter region of target genes in a heterodimeric fashion with Retinoid X Receptor (hereinafter RXR, Forman et al., Cell, 81, 687–93, 1995). The relevant physiological ligands of NR1H4 seem to be bile acids (Makishima et al., Science, 284, 1362–65, 1999; Parks et al., Science, 284, 1365–68,1999). The most potent is chenodeoxycholic acid, which regulates the expression of several genes that participate in bile acid homeostasis.
Farnesol, originally described to activate the rat ortholog at high concentration does not activate the human or mouse receptor. FXR is expressed in the liver, small intestine, colon, ovary, adrenal gland and kidney. Like LXR-α, NR1H4 is involved in autocrine signaling.
FXR is proposed to be a nuclear bile acid sensor. As a result, it modulates both, the synthetic output of bile acids from the liver and their recycling in the intestine (by regulating bile acid binding protein). Upon activation (e.g. binding of chenodeoxycholic acid), it influences the conversion of dietary cholesterol into bile acids by inhibiting the transcription of key genes which are involved in bile acid synthesis such as CYP7A1 or in bile acid transport across the hepatocyte membranes such as the bile acid transporters BSEP (Bile Salt Export Pump) and NTCP (Na-Taurocholate Co-Transporter). This seems to be a major mechanism of feedback regulation onto bile acid synthesis. Moreover, NR1H4 seems to be the crucial receptor for maintaining bile acid homeostasis within the hepatocyte and therefore might be an appropriate drug target to treat diseases that result from impaired bile acid production, impaired export into the bile canaliculi or impaired bile flow in general such as cholestatic conditions. Loss of function of NR1H4 results in major changes in bile acid homeostasis on the organism level (Lu, et al., Mol Cell. (2000) 6(3):507–15; Goodwin, et al., Mol Cell. (2000) 6(3):517–26; Sinal, et al., Cell (2000) 15; 102(6):731–44).
The synthetic compounds, 1,1-bisphosphonate esters, appear to display a number of similar activities to the two identified prototypes of natural FXR agonists, farnesol, and chenodeoxycholic acid. Like farnesol, the 1,1-bisphosphonate esters increase the rate of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase degradation and like bile acids they induce the expression of the Intestinal Bile Acid Binding Protein (I-BABP) and repress the cholesterol 7 α-hydroxylase gene. Certain 1,1-bisphosphonate esters also bind to FXR. (Niesor et al., Curr Pharm Des, 7(4):231–59, 2001). That means that activation of FXR could lead to opposing effects such as lowering the rate of cholesterol synthesis by increasing degradation of HMG-CoA Reductase and increasing the cholesterol pool by inhibition of cholesterol degradation into bile acids. The FXR agonist chenodeoxycholic acid does not change cholesterol and lipoprotein levels significantly in patients, although a repression of bile acid synthesis as well as a decreased HMG-CoA reductase activity was observed (Einarsson et al., Hepatology, 33(5), 1189–93, 2001) confirming that cellular cholesterol synthesis and degradation are controlled by numerous regulatory loops including the coordinate regulation of HMGCoA reductase and cholesterol 7α-hydroxylase and that compounds modulating FXR acitvity might have different effects on blood lipid parameters.
In the course of functional analysis of certain 1,1-bisphosphonate esters, it was shown that these compounds, which are known to bind to FXR also induce apoptosis in a variety of cell types, similar to the isporenoids farnesol and geranylgeraniol, which are also known as weak FXR binders (Flach et al., Biochem Biophys Res Com, 270, 240–46, 2000).
To date only very few compounds have been described which bind the NR1H4 receptor and thus show utility for treating diseases or conditions which are due to or influenced by said nuclear receptor (Maloney at al., J Med Chem, 10; 43(16):2971–4, 2000).
It is currently believed that FXR agonists might be useful to treat cholestatic conditions because they result in an upregulation of bile acid transport activity across the canalicular hepatocyte membrane (Plass, et al., Hepatology. (2002) 35(3):589–96; Willson, et al., Med Res Rev. (2001) 21(6):513–22). In contrast, it is believed that compounds that act as FXR antagonists or at least as mixed agonists/antagonists might reduce total serum cholesterol (Urizar, et al., Science (2002) 31; 296(5573):1703–6).
It is thus an object of the present invention to provide for novel NR1H4 binding compounds. It is thus an object of the present invention to provide for compounds which by means of binding the NR1H4 receptor act as agonist or antagonist or mixed agonist/antagonist of said receptor and thus show utility for treating diseases or conditions which are due to or influenced by said nuclear receptor.
It is further an object of the invention to provide for compounds which may be used for the manufacture of a medicament for the treatment of cholesterol or bile acid associated conditions or diseases. In a preferred embodiment of the invention, it is an object of the invention to provide for cholesterol lowering or anti-cholestatic compounds. It is also an object of the invention to provide for compounds that may be used for the manufacture of anticancer medicaments or apoptosis-inducing medicaments in general.
It is further an object of the invention to provide for compounds which are orally available and can be used for an oral treatment of the diseases mentioned afore.